Hybridoma generation for diagnostic applications

IHC and ICC are antibody-based methods for staining specific cells (ICC) or tissues (IHC) samples. Together they represent a powerful microscopy-based technique providing a clear visual and spatial output at the tissue or cellular level of specific markers and how they correlate with different cell types, cellular compartments, or biological states.

Antibodies used in IHC or ICC assays are labeled with chromogenic reagents such as horseradish peroxidase (HRP) which then require a chemical substrate to produce a color change, making the samples easy to visualize under a light microscope. These assays are also amenable to multiplexing (the use of several enzyme reporter labels to produce different colors for different antigens).

Conventional IHC and ICC assays often use formalin-fixed paraffin-embedded (FFPE) tissues that preserve the histological morphology but can mask important epitopes. It is possible to partially revert the chemical crosslinking in FFPE tissues by using antigen retrieval protocols. However, these protocols need to be optimized for each specific tissue and antigen.

Alternatively, frozen tissues can be used to minimize this issue. But, in both cases, the conformation of target epitopes is expected to change during sample treatment, which makes antibody validation in the specific cell or tissue type vital to ensure the success of IHC/ICC experiments.

WC is a widely used method to separate and detect proteins. It involves transferring (also known as blotting) proteins, previously separated by electrophoresis, from a polyacrylamide gel to a nitrocellulose membrane for visualization. The membrane is then blocked and marker-specific antibodies (labeled with chromogenic, fluorogenic, or chemiluminescent substrates) are added for protein imaging.

This immunoassay is typically performed in denaturing conditions meaning that the secondary and tertiary structures of the desired marker are lost. The development of antibodies for these applications needs to take this loss of native structure into account. More importantly, it remains vital to validate antibodies for WB in denaturing conditions to ensure they detect only the marker of interest.

Despite its enhanced sensitivity, WB assays remain complex and labor-intensive. For this reason, nowadays they are mostly used to confirm the results obtained by other techniques in preclinical and clinical diagnostics.

IP is a popular technique to capture and concentrate proteins from complex mixtures. It allows the enrichment of specific markers, which is particularly useful when dealing with low-abundance proteins.

Besides allowing the study of a protein outside its original environment, IP can be used to study the interaction of the target protein with other molecules (proteins, DNA, RNA, cells, etc.), as these complexes tend to co-precipitate. Additional reagents may be used to stabilize these complexes prior to precipitation, thus enhancing the usefulness of the assay for the study of important interactions in the context of disease.

This technique is often used in tandem with mass spectrometry providing a powerful method for measuring and identifying low-abundance proteins in complex samples. Thus, antibodies intended for IP use need to be carefully validated in the test conditions to ensure only the target protein or complex is efficiently recovered from specific samples.

Fluorescent-labeled antibodies are the crucial reagents of IF methods, a specific type of immunostaining approach. In its essence, IF is similar to IHC/ICC, thus it can be used to detect and visualize a protein of interest on fixed (FFPE) or frozen tissues (immunohistofluorescence) or particular cell types (immunocytofluorescence).

The difference lies in how the samples are visualized. For instance, IHC/ICC assays rely on an enzymatic reporter and a substrate to produce a color change, while IF assays require only a fluorescent label and the proper lasers to produce a signal. In both cases, multiplexing (detecting several antigens) is possible provided that the fluorochromes or enzymatic reporters emit non-overlapping signals.

Developing antibodies for this approach can be especially challenging since these reagents need to be highly specific to the target and, at the same time, cause minimal background noise (minimal off-site binding). Moreover, when different antibody conjugates are used in the same assay, it is even more important to validate the panel or cocktail of the different antibodies to ensure the data generated by IF experiments are easily interpreted.

These assays have been quickly gaining ground over classical immunoassays for the fast detection of disease markers in liquid samples (e.g. fluids, blood, plasma, etc.). Antibodies used in flow cytometry are typically used to detect low abundance markers (diagnostics) or determining the efficiency of specific treatments, substantially aiding the efforts of developing targeted or personalized therapeutic approaches.

These immunoassays can be performed with fixed or unfixed samples; moreover, they typically employ fluorescence-labeled antibodies in multiplexing conditions. Due to its enhanced sensitivity, off-target binding can become problematic in flow cytometry. For this reason, validation should foresee potential issues caused by different sample types, cell fixation protocols, and multiplexing conditions.

ELISA is a plate-based immunoassay for the detection of markers in complex samples. Several ELISA formats are currently employed in clinical diagnostics depending on the abundance of the target. Generally, ELISA antibodies are tagged with enzymatic labels (similar to IHC/ICC applications) and these assays can be designed for antigen detection or quantification.

Of all ELISA formats, sandwich ELISA has become the most useful and, consequently, the most challenging to develop. Sandwich ELISA employs two primary antibodies binding non-overlapping epitopes of a given antigen. Similar to all other immunoassays, the validation of antibodies for ELISA should consider the type of samples and detection conditions to be used.

• Hybridoma production for therapeutic applications
• Hybridoma cell culture
• Small molecule antibodies
• FAQs about hybridoma production